Preparation of 11alpha, 17alpha-dihydroxyprogesterone



United States Patent Cfice 3,011,951 Patented Dec. 5, 1961 3,011,951PREPARATION OF 110:,17ot-DII'IYDR0XY- PRQGESTERONE Herbert C. Murray,Barry Township, Barry County, and Lester M. Reineke, Portage Township,Kalamazoo County, Mich, assignors to The Upjohn Company, Kalamazoo,Mich, a corporation of Delaware No Drawing. Filed Nov. 23, 1960, Ser.No. 71,167 Claims. (Cl. 195-51) The present invention relates to a novelprocess for the oxidation of steroids, and more particularly to 17a-l1Y-droxylation of lla-hydroxyprogesterone. The process of the presentinvention comprises subjecting lla-hydroxyprogesterone to theoxygenating activity of the fungus Sepedonium ampullosporum to introducea 17a-hydroxy group.

Culture of the fungus, in accordance with this invention, is in or on amedium favorable to the development of the fungus. Solid media may beutilized, but the preferred media are those which permit quantity growthunder aerobic conditions. Moist solid particulate media, such as bran,cereal grains, cereal grits, wood chips, shavings, sawdust, cornhusks,fibrousmaterial, such as copra, chestnuts, or lupine seeds may be used.These can be extracted with alcohol, ether or other organic solvents, toremove objectionable contaminants and growth inhibitors prior tofermentation. The carriers may optionally contain added growth factorsand nutrients and may be used in layers or trays with or withoutauxiliary aeration, in towers as in the vinegar process or underconditions of agitation as for example by tumbling in a rotating drum.Liquid media, illustratively brewers wort, are vell adapted to use underaerobic layer or more especially aerobic submerged fermentationconditions. Suitably the media should contain sources of availablecarbon, nitrogen and minerals although of course there can besignificant growth and development under less than optimum conditions.

Available carbon may be from carbohydrates, starches, gelatinizedstarches, dextrin, sugars, molasses as of cane, beet and sorghum,dextrose, glucose, fructose, mannose, galactose, maltose, sucrose,lactose, pentoses, amino acids, peptones or proteins. Carbon dioxide,glycerol, alcohols, acetic acid, sodium acetate, citric acid, sodiumcitrate, lower fatty acids, higher fatty acids, or fats are illustrativeof other materials which provide assimilable carbon for the energyrequirements of the fungus. Mixtures of various carbon sources aresometimes advantageous.

Nitrogen in assimilable form can be provided by soluble or insolublevegetable or animal proteins, soybean meal, lactalbumin, casein, eggalbumin, peptones, polypeptides or amino acids, urea, ammonium salts,ammonia trapped on base exchange resins or on zeolites, ammoniumchloride, sodium nitrate, potassium nitrate, or morpholine. Whey,distillers solubles, corn steep liquor, or yeast extract have beenuseful.

As mineral constituents the media or menstruum can contain, naturallypresent or added, available aluminum, calcium, chromium, cobalt, copper,gallium, iron, magnesium, molybdenum, potassium, scandium, uranium andvanadium. Sulfur can be provided by sulfates, alkyl sulfonates,sulfoxylates, sulfamates, sulfinates, free sulfur, hyposulfite,persulfate, thiosulfate, methionine, cystine, cystein, thiamin orbiotin. Phosphorus, preferably pentavalent, suitably in a concentrationat or about 0.001 to 0.07 molar and particularly at or about 0.015 to0.02, can be present, suitably as ortho-, meta-, of pyrophosphates,salts or esters, phytin, phytic acid, phytates, glycerophosphate, sodiumnucleinate, and/or corn steep liquor, casein or ovovitellin. Boron,iodine and selenium in traces may be advantageous. Desirably boron, inthe form of boric acid or sodium borate can be present or addedespecially after germination and early growth of the fungus.

Other accessory growth factors, vitamins, auxins and growth stimulantscan be provided as needed or desired.

While solid or liquid media can be utilized, a liquid medium ispreferred as it favors mycelial growth.

Suspending or mycelial carriers such a filter earths, filter aids,finely divided cellulose, wood chips, bentonite, calcium carbonate,magnesium carbonate, charcoal, activated carbon or other suspendablesolid matter, methyl cellulose, carboxymethyl cellulose or alginates canbe added to facilitate fermentation, aeration and filtration.

Any suitable strain of Sepedonium ampullosporum can be used in thepractice of this invention. It will be illustrated below with an exampleemploying a particular strain which we prefer.

The fungus is grown on a medium suitably containing assimilablenon-steroidal carbon, illustratively carbohydrates, such as sugar orstarches; assimilable nitrogen, illustratively soluble or insolubleproteins, peptones or amino acids; and mineral constituents,illustratively phosphates and magnesium sulfate; and other artrecognized, desirable additions. The medium can desirably have a. pHbefore inoculation of between about 4 to 7 although a higher or lower pHmay be used. A pH of between about 4 and 5 ispreferred for the growth ofSepedonium ampullosporum.

Inoculation of the fungal growth-supporting medium with Sepedoniumampullosporum can be accomplished in any suitable manner. Sepedoniumampullosporum grows over a range from about 20 to about 38 C., with atemperature between about 25 to about 35 C. preferred.

The developmental period of fungal growth required before the steroid tobe fermented is exposed to the fungus does not appear to be critical.For example, the steroid can be added either before thermal or othersterilization of the medium, at the time of inoculating the medium withScpedonium ampullosporum, or at some time, for example, 24 or 48 hours,later. The steroid to be fermented can be added at any suitableconcentration although for practical reasons steroid substrate at aconcentration of about or up to about 0.6-grarn per liter or even 0.8gram per liter of medium is satisfactory and two grams per liter isoperative although higher concentration, depending on the particularsteroid, can be used with some inhibition of mycelial development. Theaddition of steroid substrate to be fermented can be accomplished in anysuitable manner especially so as to promote a large surface of contactof the steroid substrate with the fungus, such as by dispersing thesteroid substrate, either alone, with a dispersing agent, or in solutionin an organic solvent by mixing or homogenizing a steroid substrate.

with a fungal medium to form a suspension or dispersion of steroid.Either submerged or surface culture procedures can be used withfacility, although submerged culture is preferred. Alternatively,steroid fermenting enzymes of a growth of the fungus can be separatedfrom the fungus or medium, admixed with the steroid or a solution ordispersion thereof, and the mixture subjected to aerobic conditions toaccomplish fermentation of the steroid.

The temperature during the period of fermentation of the steroid can bethe some as that found suitable for fungal growth. It need be maintainedonly within such range as supports life, active growth, or the enzymeactivity of the fungus.

While any form of aerobic incubation is satisfactory for the growth ofthe selected fungus and fermentation of the steroid substrate, theefficiency of steroid fermentation is related to aeration. Therefore,aeration is usually con- Aerorbic conditions: include not only the useof air'to' introduce oxygen, but also other sources or mixturescontaining oxygen in :free or liberatable form. in using air as theaerating medium, a desirable rate of aeration is about 4 to 20millimoles and particularly about 6 millimoles of oxygen per hour perliter as determined by the method of Cooper, Fernstrom and Miller, Ind.Eng. Chem. 36, 504 (1944). Aeration is suitably modified by usingsuperatmospheric or su-batmospheric pressures, for example, 30 poundsper square inch or 10 pounds per square inch absolute. Oxygen uptake maybe facilitated by the presence of various agents such as ascorbic acid,glutamic acid, citric acid, lactic acid, tyrosine, or tryptophane.

The time required for the fermentation of steroid varies somewhat withthe procedure. When the steroid substrate is present at the time ofinoculation of the medium, periods of from 8 to 72 hours may be used.However, when the steroid is added to the fungus, after substantialaerobic growth of the fungal organism, for example, after 16 to 24 hoursat optimum temperature, the conversion of steroid substrate beginsimmediately and high yields are obtained in from 1 to 72 hours, 48 hoursbeing generally satisfactory.

After completion of the steroid fermentation, the resulting fermentedsteroid is recovered from the fermentation reaction mixture. Anespecially advantageous manner of recovering the fermented steroidinvolved extracting the fermentation reaction mixture, including thefermentation liquor and mycelia with a water-immiscible organic solventfor steroids, for example, methylene chloride, chloroform, carbontetrachloride, ethylene chloride, trichloroethylene, ether, amylacetate, benzene, and the like. The fermentation liquor and mycelia maybe separated and then separately extracted with suitable solvents. Themycelia maybe extracted with either water miscible or water-immisciblesolvents, acetone being effective. The fermentation liquor, freed ofmycelia, may be extracted with'water-immiscible solvents. The extractscan be combined, either before or after washing with an' 'oxygenatingactivity which is probably enzymatic in character. This activityisassociated With the hyphal mass or with the spores, and the describedNot-oxygenation can be accomplished by contacting culture medium, thehyphal mass, or spores with lla-hydroxyprogesterone.

According to this aspect of the invention, the fungus can be cultured asdescribed above and the hyphal mass or spores can be separated from thefungal growth-supporting medium, according to procedures well-known inthe art. The hyphal mass or spores are then resuspended in tap water,buffered distilled water or other suitable menstruum, and brought intocontact, under aerobic conditions, with lla-hydroxyprogesterone toproduce 11a, 17a-dihydroxyprogesterone.

The following example is illustrative of the process and product of theinvention.

Example.] 1 a,] 7u-dihydroxyprogesterone A medium was prepared of 20 g.of cornsteep liquor (60% solids) and g. of commercial dextrose, dilutedto 1 l. and adjusted to a pH between 4.8 and 5. 10 ml. of lard oil wasadded as an antifoam preventive, 10 1. of this sterilized medium Wasinoculated with a 72-hour vegetative growth of Sepedonium ampullosporum[Damon, Mycologia, vol. 44, pages 86-96 (1952)], a culture of which isavailable from the Northern Utilization Research and DevelopmentDivision, Peoria, Illinois, having been assigned the numericaldesignation NRRL 2877, or from the Centralibureau voor Schimmelcultures,Baarn, Holland, and incubated for 48 hours at a temperature of about 28C. using a rate'of aeration of 0.3 l. per minute at 300 r.p.m. After 48hours of agitation, a solution of 2.0 g. of lla-hydroxyprogesterone in20 ml. of dimethylformamide was added to the inoculated medium. After anadditional 48-hour period of incubation, the beer and mycelium wereseparated by filtration. The mycelium was washed with water and thewashwater was added to the beer filtrate. The thus-obtained beerfiltrate was extracted 4 times with a volume of methylene chloride equalto one-fourth the volume of the filtrate. The combined extracts werewashed with one-fourth volume of distilled water and the solvent wasremoved by distillation to give a crude residue which was dissolved inabout 25 ml. of methylene chloride and poured onto a column of Florisil(synthetic magnesium silicate) 3.3 cm. in diameter and 35 cm. high. Thecolumn was developed by eluting with 335 ml. portions (except asotherwise indicated) of solvents and the eluate fractions wereevaporated to dryness and'the Weight of the residue of each fractiondetermined, as shown in Table I.

TABLE I Fraction Solvent Weight of Residue 1 Commercial hexanes (1liter) 221 mg., oily. 2 5% Acetone in Commercial hex- 0.

anes. 3. do 312 mg., oily. 4 do 78 mg, oily. 5 10% Acetone in Commercialhex- 31 mg, oily. 6. do 89 mg, oily. 7. mg oily. 8. 15 mg. 9- 12 mg. 10.10 mg. 11- 47 mg. 12. 527 mg. 11a, 17a- 13. 385 mg. dihydrcxy- 14. 64mg. progesterone. 15- 30 mg. 16 10 mg. 17. 305 mg. 1s 140 mg. 19 11 mg.

Fractions l2, l3 and 14 of the chromatogram were combined andrecrystallized from acetone-commercial hexanes to give 0.56 g. of1la,17a-dihydroxyprogesterone having a melting point of 2l02l4 C. and

Am 242 my, 15,700

The infrared spectrum was the same as that of an authentic sample of1la,l7a-dihydroxyprogesterone.

We claim:

'1. A process for producing l1a,17a-dihydroxypr0gesterone whichcomprises aerobically subjecting Ila-hydroxyprogesterone to theoxygenating activity of Sepedonium ampullosporum.

2. A process for the production of :,17a-dihYdI'OXY- progesterone whichcomprises aerobically contacting llahydroxyprogesterone with Sepedoniumampullosporum.

3. A process for the production of lla,l7: x-dihydroxyprogesterone whichcomprises contacting lloc-hYdI'OXY- progesterone under aerobicconditions in the presence of a nutrient medium with Sepedoniumampullosporum.

4. A process for the production of l1u,17a-dihydroxyprogesterone whichcomprises growing Sepedonium ampullosporum under aerobic conditions inthe presence of a nutrient medium and llu-hydroxyprogesterone.

5. A process for the production of 11a,17a-dihydroxyprogesterone whichcomprises growing Sepedonium ampullosporum under aerobic conditions, inthe presence carbon and lla-hydroxyprogesterone and recovering theresulting l1a,17a-dihydroxyprogesterone.

6. A process for the production of 11a,17m-dihydroxyprogesterone whichcomprises growing Sepedonium ampullosporum under aerobic conditions withagitation in the presence of a nutrient medium containing assimilablecarbohydrate, nitrogen and phosphorus and 11cchydroxyprogesterone andrecovering 11a,17a-dihydroxyprogesterone.

7. A process for the production of 11a,17a-dihydroxyprogesterone whichcomprises growing Sepedonz'um ampullosporum under aerobic conditions andwith agitation in the presence of a nutrient medium containingassimilable carbohydrate, nitrogen, phosphorus and11ahydroxyprogesterone and separating the thus-produced 1 1oz,17a-dihydroxyprogesterone.

8. A process for the production of 11a,17a-dihydroxyprogesterone whichcomprises growing Sepedonium ampullosporum under aerobic, submergedconditions in the presence of a nutrient medium containing assimilablecarbohydrate, nitrogen, and phosphorus and lla-hydroxy- References Citedin the file of this patent UNITED STATES PATENTS Murray et a1. Feb. 16,1960 Dulaney et a1. Jan. 31, 1961

3. A PROCESS FOR THE PRODUCTION OF 11A,17A-DIHYDROXYPROGESTERONE WHICHCOMPRISES CONTACTING 11A-HYDROXYPROGESTERONE UNDER AEROBIC CONDITIONS INTHE PRESENCE OF A NUTRIENT MEDIUM WITH SEPEDONIUM AMPULLOSPORUM.